Compositions and methods for inhibiting angiogenesis

ABSTRACT

The invention provides for methods of reducing or inhibiting angiogenesis, tumor growth and endothelial cell proliferation by the administration of compositions containing fragments, conformations, biological equivalent, or derivatives of antithrombin III. The invention also provides for pharmaceutical compositions comprising a fragment, conformation, biological equivalent, or derivative of antithrombin III and methods of identifying novel inhibitors of tumor growth, endothelial cell proliferation, and/or angiogenesis. The invention also relates to compositions and methods for altering angiogenesis in a mammal, as well as to methods of treatment for disorders associated with angiogenesis (e.g., cancer).

[0001] This application claims benefit of U.S. Provisional ApplicationsSerial Nos. 60/103,526 filed Oct. 8, 1998 and 60/116,131 filed Jan. 15,1999, the disclosures of which are hereby incorporated by reference.

[0002] The invention was supported, in whole or in part, by grantCA45548 from the National Institutes of Health. The Government hascertain rights in the invention.

[0003] Blood vessels are constructed by two processes: vasculogenesis,whereby a primitive vascular network is established during embryogenesisfrom multipotential mesenchymal progenitors; and angiogenesis, in whichpreexisting vessels send out capillary sprouts to produce new vessels.Endothelial cells are centrally involved in each process. They migrate,proliferate and then assemble into tubes with tight cell-cellconnections to contain the blood (Hanahan, Science 277:48-50 (1997)).Angiogenesis occurs when enzymes, released by endothelial cells, andleukocytes begin to erode the basement membrane, which surrounds theendothelial cells, allowing the endothelial cells to protrude throughthe membrane. These endothelial cells then begin to migrate in responseto angiogenic stimuli, forming off-shoots of the blood vessels, andcontinue to proliferate until the off-shoots merge with each other toform the new vessels.

[0004] Normally angiogenesis occurs in humans and animals in a verylimited set of circumstances, such as embryonic development, woundhealing, and formation of the corpus luteum, endometrium and placenta.However, aberrant angiogenesis is associated with a number of disorders,including, tumor metastasis. In fact, it is commonly believed that tumorgrowth is dependent upon angiogenic processes. Thus, the ability toincrease or decrease angiogenesis has significant implications forclinical situations, such as wound healing (e.g., graft survival) orcancer therapy, respectively.

[0005] Antithrombin or Antithrombin III (AT3) is a single chainglycoprotein involved in the coagulation process. It is synthesizedprimarily in the liver with a signal peptide of 32 amino acids necessaryfor its intracellular transport through the endoplasmic reticulum; thepeptide is then cleaved prior to secretion. Mourey et al, Biochimie72:599-608 (1990).

[0006] AT3 is a member of the serpin family of proteins and functions asan inhibitor of thrombin and other enzymes involved in the clottingcascade. As used herein, the active native intact form of AT3 isdesignated the S (stressed) form (S-AT3). S-AT3 forms a tight bindingcomplex with thrombin (markedly enhanced by the presence of heparin) andother enzymes (not all serpins have heparin affinity).

[0007] S-AT3 can be cleaved to the relaxed (R)-conformation (R-AT3) by avariety of enzymes, including thrombin. Evans et al., Biochemistry31:1262912642 (1992). For example, it has been thought that thrombinbinds to a reactive C-terminal loop of AT3 and the resultant complexslowly dissociates releasing thrombin and cleaving off the C-terminalloop of inactive AT3, resulting in R-AT3. R-AT3 is unable to bindthrombin and has a conformation that is quite different from that ofS-AT3. The role of R-AT3 had only been known to facilitate hepaticclearance of the molecule.

[0008] Other forms of AT3, such as L-AT3, which is the group of forms ofATIII that includes both the latent form and the locked form, aresimilar in conformation to R-AT3, and are also known in the art. Carrellet al., Nature 353, 576-578 (1991); Wardell et al., Biochemistry 36,13133-13142 (1997). L-AT3, for example, can be produced by limiteddenaturing and renaturing the AT3 protein under specific temperatureconditions, e.g., with guanidium chloride.

[0009] Prior to the present invention, AT3 was not known to beassociated with angiogenesis. The present invention is, in oneembodiment, drawn to a fragment, conformation, derivative or biologicalequivalent of AT3 that inhibits endothelial cell proliferation,angiogenesis and/or tumor growth in vivo.

[0010] In one embodiment, the invention relates to a method ofinhibiting tumor growth by delivering or administering a compositioncomprising a fragment, conformation, biological equivalent, orderivative of AT3. In a preferred embodiment, the fragment,conformation, biological equivalent, or derivative of AT3 is chosen fromthe L form of AT3, the R form of AT3 and fragments that include theactive sites of the L form of AT3 and/or the R form of AT3. Thefragment, conformation, biological equivalent, or derivative of AT3 mayalso be chosen from a synthesized fragment of AT3 that inhibits tumorgrowth, conformational variations of other serpins that inhibit tumorgrowth, an aggregate form of AT3 that inhibits tumor growth, or a fusionprotein of AT3 that inhibits tumor growth. The composition may furthercomprise a physiologically acceptable vehicle.

[0011] The invention further relates to a method of inhibitingendothelial cell proliferation comprising delivering or administering acomposition comprising a fragment, conformation, biological equivalent,or derivative of AT3. In a preferred embodiment, the fragment,conformation, biological equivalent, or derivative of AT3 is chosen fromthe L form of AT3, the R form of AT3 and fragments that include theactive sites of the L form of AT3 and/or the R form of AT3. Thefragment, conformation, biological equivalent, or derivative of AT3 mayalso be chosen from a synthesized fragment of AT3 that inhibitsendothelial cell proliferation, conformational variations of otherserpins that inhibit endothelial cell proliferation, an aggregate formof AT3 that inhibits endothelial cell proliferation, or a fusion proteinof AT3 that inhibits endothelial cell proliferation. The composition mayfurther comprise a physiologically acceptable vehicle.

[0012] The invention also relates to a method of reducing or inhibitingangiogenesis comprising delivering or administering a compositioncomprising a fragment, conformation, biological equivalent, orderivative of AT3. In a preferred embodiment, the fragment,conformation, biological equivalent, or derivative of AT3 is chosen fromthe L form of AT3, the R form of AT3 and fragments that include theactive sites of the L form of AT3 and/or the R form of AT3. Thefragment, conformation, biological equivalent, or derivative of AT3 mayalso be chosen from a synthesized fragment of AT3 that reducesangiogenesis, conformational variations of other serpins that reduceangiogenesis, an aggregate form of AT3 that reduces angiogenesis, or afusion protein of AT3 that reduces angiogenesis. The composition mayfurther comprise a physiologically acceptable vehicle.

[0013] In another embodiment, the invention pertains to a method foridentifying an inhibitor of tumor growth or an agent that reduces tumorgrowth, comprising the steps of inoculating an animal with anappropriate innoculum of tumor cells in each of two suitable inoculationsites; identifying inhibition of growth of a tumor, known as thesubordinate tumor, at one inoculation site with concomitant growth of atumor, known as the dominant tumor, at the other inoculation site;isolating cells from the dominant tumor; and purifying a component whichinhibits endothelial cell proliferation and/or angiogenesis from theisolated cells. For example, the component may be purified fromconditioned media from the cells. In one embodiment of the invention,the tumor cells are derived from tumors selected from the groupconsisting of small cell lung cancers and hepatocellular carcinomas. Ina particular embodiment, the inoculation sites are the flanks of theanimal. In one embodiment the inhibitor of tumor growth is an inhibitorof endothelial cell proliferation. In another embodiment the inhibitorof tumor growth is an inhibitor of angiogenesis. In a further embodimentof the invention, the method further comprises a step of selecting foran animal in which inhibition of the growth of the subordinate tumor bythe dominant tumor is substantially complete.

[0014] The invention further relates to a method of inhibiting tumorgrowth comprising delivering or administering an inhibitor of tumorgrowth identified by the methods described herein to a mammal. In apreferred embodiment the inhibitor of tumor growth is a fragment,conformation, biological equivalent, or derivative of AT3.

[0015] It is also within the practice of the invention to use a similarmethod to identify an agent that reduces or an inhibitor of angiogenesisand/or endothelial cell proliferation. Such a method would alsocomprise-the steps of inoculating an animal with an appropriate inoculumof tumor cells in each of two suitable inoculation sites; identifyinginhibition of growth of a tumor, known as the subordinate tumor, at oneinoculation site with concomitant growth of a tumor, known as thedominant tumor, at the other inoculation site; isolating cells from thedominant tumor; and purifying a component which inhibits endothelialcell proliferation and/or angiogenesis from the isolated cells. Thecomponent may be purified from conditioned media from the cells and in aparticular embodiment, the inoculation sites are the flanks of theanimal.

[0016] The invention further relates to a method of reducing orinhibiting angiogenesis and/or endothelial cell proliferation comprisingdelivering or administering an inhibitor of angiogenesis and/orendothelial cell proliferation identified by the methods describedherein to a mammal. In a preferred embodiment the inhibitor ofangiogenesis and/or endothelial cell proliferation is a fragment,conformation, biological equivalent, or derivative of AT3.

[0017] The invention also relates to a method of treating a disordermediated by angiogenesis comprising delivering or administering acomposition comprising a fragment, conformation, biological equivalent,or derivative of AT3 in an amount effective to reduce angiogenesis to amammal. In a preferred embodiment the fragment, conformation, biologicalequivalent, or derivative of AT3 is chosen from the L form of AT3, the Rform of AT3 and fragments that include the active sites of the L form ofAT3 and/or the R form of AT3. The fragment, conformation, biologicalequivalent, or derivative of AT3 may also be chosen from a synthesizedfragment of AT3 that reduces angiogenesis, conformational variations ofother serpins that reduce angiogenesis, an aggregate form of AT3 thatreduces angiogenesis, or a fusion protein of AT3 that reducesangiogenesis. The composition may further comprise a physiologicallyacceptable vehicle.

[0018] The invention also relates to a method of treating a disordermediated by endothelial cell proliferation comprising delivering oradministering a composition comprising a fragment, conformation,biological equivalent, or derivative of AT3 in an amount effective toinhibit endothelial cell proliferation to a mammal. In a preferredembodiment the fragment, conformation, biological equivalent, orderivative of AT3 is chosen from the L form of AT3, the R form of AT3and fragments that include the active sites of the L form of AT3 and/orthe R form of AT3. The fragment, conformation, biological equivalent, orderivative of AT3 may also be chosen from a synthesized fragment of AT3that inhibits endothelial cell proliferation, conformational variationsof other serpins that inhibit endothelial cell proliferation, anaggregate form of AT3 that inhibits endothelial cell proliferation, or afusion protein of AT3 that inhibits endothelial cell proliferation. Thecomposition may further comprise a physiologically acceptable vehicle.

[0019] The invention also relates to a method of enhancing angiogenesiscomprising delivering or administering a composition comprising aneffective amount of an antagonist of a fragment, conformation,biological equivalent, or derivative of AT3 wherein the fragment,conformation, biological equivalent, or derivative of AT3 reducesangiogenesis to a mammal. This method can be used, for example, in woundhealing and assisted reproduction techniques as well as in coronaryartery surgery and the revascularization/collateralization of peripheralvascular vessels. The composition may further comprise a physiologicallyacceptable vehicle.

[0020] The invention also relates to a method of enhancing endothelialcell proliferation comprising delivering or administering a compositioncomprising an effective amount of an antagonist of a fragment,conformation, biological equivalent, or derivative of AT3 wherein thefragment, conformation, biological equivalent, or derivative of AT3inhibits endothelial cell proliferation to a mammal. The composition mayfurther comprise a physiologically acceptable vehicle.

[0021] Another embodiment of the invention is a kit for detecting thepresence of a fragment, conformation, biological equivalent, orderivative of AT3. The kit may contain primary reagents suitable fordetecting the presence of the fragment, conformation, biologicalequivalent, or derivative of AT3 and optional secondary agents suitablefor detecting the binding of the primary reagent to the fragment,conformation, biological equivalent, or derivative of AT3. In apreferred embodiment, the fragment, conformation, biological equivalent,or derivative of AT3 is the L form of bovine AT3, the R form of bovineAT3, the L form of human AT3, or the R form of human AT3.

[0022] The invention provides for direct administration of the fragment,conformation, biological equivalent, or derivative of AT3, along withthe use of the fragment, conformation, biological equivalent, orderivative of AT3 with or without physiologically acceptable vehicles,including but not limited to viral vectors including adenoviruses,lipids and any other methods that have been employed in the art toeffectuate delivery of biologically active molecules.

[0023] The invention also provides for the production of a fragment,conformation, biological equivalent, or derivative of AT3 in vivo by thedelivery of an enzyme. It is also within the practice of the inventionto produce a fragment, conformation, biological equivalent, orderivative of AT3 in vivo by the delivery of a composition thateffectuates a conformational change in a serpin.

[0024] The invention also relates to pharmaceutical compositionscomprising a fragment, conformation, biological equivalent, orderivative of AT3. The composition may be effective for inhibiting tumorgrowth, angiogenesis, and/or endothelial cell proliferation. In oneembodiment, an anti-angiogenic pharmaceutical composition comprises apurified form of AT3 that reduces angiogenesis. In a preferredembodiment the purified form of AT3 is the L form or R form of AT3 or afragment or sequence which includes the active site or region of the Lform or R form of AT3. The composition may further comprise aphysiologically acceptable vehicle. The fragment, conformation,biological equivalent, or derivative of AT3 may be an active ingredientin a pharmaceutical composition that includes carriers, fillers,extenders, dispersants, creams, gels, solutions and other excipientsthat are common in the pharmaceutical formulatory arts.

[0025] The invention also provides for a method of delivering oradministering a composition comprising a fragment, conformation,biological equivalent, or derivative of AT3 by any methods that havebeen employed in the art to effectuate delivery of biologically activemolecules, including but not limited to, administration of anaerosolized solution, intravenous injection, orally, parenterally,topically, or transmucosally.

[0026] The invention also provides for a pharmaceutical composition thatcomprises compositions to facilitate delivery of therapeuticallyeffective amounts of the fragment, conformation, biological equivalent,or derivative of AT3. The pharmaceutical compositions of the inventionmay be formulated to contain one or more additional physiologicallyacceptable substances that stabilize the compositions for storage and/orcontribute to the successful delivery of the fragment, conformation,biological equivalent, or derivative of AT3.

[0027] Additional features and advantages of the invention will be setforth in the description which follows, and, in part, will be apparentfrom the description, or may be learned by the practice of theinvention. The objectives and other advantages of the invention will berealized and attained by the compounds and methods particularly pointedout in the written description and claims hereof as well as the appendeddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1a. shows three conformations of AT3. b. (SEQ ID NO: 1) showsthe amino acid sequence of human AT3 and the pancreatic elastasecleavage site of the c-terminal reactive loop.

[0029]FIG. 2 shows the sequence data and schematic of bovine AT3 andaaAT3. N-terminal sequences were determined by automated Edmandegradation on a PE/ABD Procise 494cLC protein sequencer (Foster City,Calif.) with high sensitivity phenylthiohydantoin amino acid detectionby capillary HPLC. Sequence library searches and alignments wereperformed against combined GenBank, Brookhaven Protein, SWISS-PROT, andPIR databases.

[0030]FIG. 3 is a graph showing the effects of intact (S), cleaved (R)and (L) conformations of bovine AT3 on capillary endothelial cellproliferation.

[0031]FIG. 4 is a graph showing the effects of intact (S), cleaved (R)and (L) conformations of human AT3 on capillary endothelial cellproliferation.

[0032]FIG. 5a. is a graph showing the results of treatment with humanS-AT3, human R-AT3, human L-AT3 and bovine R-AT3 in mice implanted witha human neuroblastoma. The mean tumor volume and standard error for mice(n=4/group) is shown. Tumor volume was determined using the formulawidth²×length×0.52. The experiment was terminated and mice sacrificedand autopsied when control mice began to die or experience morbidity. b.Photograph of representative mice from each group on the 12^(th) day oftreatment. Arrows indicate tumors that have partially regressed.

[0033]FIG. 6 shows the treatment of human neuroblastoma with latent orlocked human antithrombin III. Mean tumor volume and standard error(n=5/group).

[0034]FIG. 7 depicts a model of concomitant resistance. a. By selectivein vivo passage, a variant of NCI-H69; small cell lung cancer wasdeveloped in which a primary flank tumor completely suppresses thegrowth of a second implant on the opposite flank (arrows). b. In anothervariant of the small cell lung cancer line that does not produce aaAT3,no evidence of concomitant resistance is observed.

[0035]FIG. 8 Purification of aaAT3 from the conditioned media ofNCI-H69i spheroids. a. Purification scheme. b. SDS-PAGE (reduced) ofintact AT3 (lane 1) and aaAT3 (lane 2) purified from H69i conditionedmedia. The B-chain of the cleaved form is indicated with an arrow.

DETAILED DESCRIPTION OF THE INVENTION

[0036] As previously discussed, aberrant angiogenesis is associated witha number of disorders including tumor metastasis. Additionally,endothelial cells are associated with angiogenesis. In the presentinvention, fragments, conformations, biological equivalents, orderivatives of AT3 may exhibit anti-angiogenic and anti-tumor activity.For example, the R and L forms of AT3 are endothelial cell specific.Thus, this invention provides for methods of reducing or inhibitingangiogenesis, tumor growth and endothelial proliferations usingfragments, conformations, biological equivalents, or derivatives of AT3.The invention also provides for methods of identifying inhibitors oftumor growth, endothelial cell proliferation, and/or angiogenesis. Theinvention also provides for pharmaceutical compositions comprising afragment, conformation, biological equivalent, or derivative of AT3. Theelements of the invention will now be discussed.

[0037] AT3 can be derived from any organism which produces the proteinin nature. In a particular embodiment the organism is bovine or human.The amino acid sequence of bovine AT3 is available under GenBankAccession No. 1168462, and the amino acid sequence of human AT3 isavailable under GenBank Accession No. 113936 (SEQ ID NO:1).

[0038] AT3 can be isolated from body fluids such as serum, ascites andurine. AT3 can also be synthesized chemically or biologically, such asby cell culture or recombinant technology, or produced transgenically.Similarly, the particular portions and conformations of AT3, which arethe subject of this invention, can be isolated from natural sources,produced transgenically, or can be chemically or biologicallysynthesized, such as by guanidine treatment or in vitro cleavage of AT3.Recombinant techniques known in the art include, but are not limited to,gene amplification from DNA using polymerase chain reaction (PCR), geneamplification from RNA using reverse transcriptase PCR and NASBA(nucleic acid sequence based amplifications).

[0039] The particular portions and conformations of AT3 or itsbiological equivalents, which are the subject of this invention, mayalso be produced by the use of an enzyme (e.g. elastase) in vivo. Forexample, an enzyme may be used in vivo, with or without plasma or nativeAT3 to serve as an additional substrate, to produce a fragment,conformation, biological equivalent, or derivative of AT3 that inhibitsendothelial cell proliferation, angiogenesis and/or tumor growth.

[0040] AT3 is a member of the serpin family of proteins and functions asan inhibitor of thrombin and other enzymes involved in the clottingcascade. Serpins are a family of proteins that function as serineprotease inhibitors. As used herein, the active native intact form ofAT3 is designated the S (stressed) form (S-AT3). S-AT3 (FIG. 1A) isreferred to as the “stressed” form of AT3 due to the fact that it is ametastable conformation with the reactive center loop (RCL) extended.This is the active form of AT3 in terms of inhibition of thrombin andother serine proteases.

[0041] S-AT3 can be cleaved to the relaxed (R)-conformation (R-AT3) by avariety of enzymes, including thrombin. R-AT3 (FIG. 1A) is referred toas the “relaxed” form due to the fact that the RCL has been cleaved byone of several proteases, including thrombin and elastase. This resultsin the insertion of the N-terminal half of the loop as a sixth strandinto the A-beta sheet of AT3 to give a much more stable conformationthan for the S-AT3 form. This form is no longer active as an inhibitorof serine proteases. For example, thrombin binds to a reactiveC-terminal loop of AT3 and the resultant complex slowly dissociatesreleasing thrombin and cleaving off the C-terminal loop of inactive AT3,resulting in R-AT3. In particular, R-AT3 can be generated by enzymecleavage of S-AT3 between Arg₃₉₃ and Ser₃₉₄ (human AT3 numbering). Theamino acid sequence of human AT3 and the pancreatic elastase cleavagesite of the c-terminal reactive loop are shown in FIG. 1B. (For bovineR-AT3, the cleavage site is between Ser₃₈₆ and Thre₃₈₇ as shown in FIG.2.) The cleaved AT3 consists of disulfide-bonded A- and B-chains and isunable to bind thrombin. The cleavage occurs spontaneously even at coldtemperatures resulting in the R form of AT3. Enzymes suitable for thiscleavage include, but are not limited to, pancreatic elastase andthrombin. R-AT3 is unable to inhibit thrombin and has a conformationthat is quite different from that of S-AT3. The role of R-AT3 had onlybeen known to facilitate hepatic clearance of the molecule.

[0042] L-AT3 (FIG. 1A) is a group of forms of AT3 that includes the boththe latent form and the locked form. These forms are structurallysimilar to the R-AT3 form in that all or part of the N-terminal half ofthe RCL has been inserted as a sixth strand into the A-beta sheet ofAT3, resulting in a more stable conformation that is no longer active asa serine protease inhibitor. In the case of the “L-forms” however, thereis no cleavage of the loop. The latent conformation is a monomericL-form, while the locked conformation also includes dimers andoligomers, which are formed by insertion of the RCL from one AT3molecule into the A-beta sheet of another.

[0043] Surprisingly, it has been determined that certain conformationsof AT3 reduce angiogenesis, endothelial cell proliferation, and tumorgrowth. (As used herein, endothelial cell proliferation also includesendothelial cell migration and tube formation.) For example, R-AT3 haspotent anti-angiogenic and anti-tumor activity which is not found inS-AT3. Additionally, the stable locked and latent forms (L-AT3) ofS-AT3, which are substantially similar in conformation to R-AT3, alsoreduce or inhibit angiogenesis and tumor growth in vivo and areendothelial cell specific in vitro. The invention relates to methods ofinhibiting endothelial cell proliferation, angiogenesis and/or tumorgrowth in a mammal comprising delivering or administering to the mammala composition comprising a fragment, conformation, biologicalequivalent, or derivative of AT3, including but not limited to L-AT3 andR-AT3, and an optional physiologically acceptable vehicle. As describedherein, fragments conformations, derivatives and biological equivalentsof AT3 include, but are not limited to: other serpins and theirconformational variations; fragments; conformations; aggregate forms;and fusion proteins; which are active as inhibitors of angiogenesis,endothelial cell proliferation and/or tumor growth. The invention alsorelates to a method of treating a disorder mediated by angiogenesis orendothelial cell proliferation comprising administering a compositioncomprising a fragment, conformation, derivative or biological equivalentof AT3, including but not limited to L-AT3 and R-AT3, and an optionalphysiologically acceptable vehicle to a mammal. The invention furtherrelates to a method of treating cancer comprising administering acomposition comprising an effective amount of a fragment, conformation,derivative or biological equivalent of AT3, including but not limited toL-AT3 and R-AT3, and an optional physiologically acceptable vehicle to amammal.

[0044] The invention also relates to a method of enhancing angiogenesisor endothelial cell proliferation comprising administering a compositioncomprising an effective amount of an antagonist of AT3, e.g., anantagonist of S-AT3, an antagonist of R-AT3 or an antagonist of L-AT3 toa mammal. For example, this method can be useful in the treatment ofabnormal ovulation, menstruation and placentation, and vasculogenesis,such as in tissue repair, wound healing and tissue grafting.

[0045] The fragments, conformations, derivatives, and biologicalequivalents of AT3 having anti-angiogenic properties, that inhibit theproliferation of endothelial cells, and/or have anti-tumor activity aredescribed herein. These AT3 fragments, conformations, derivatives andbiological equivalents are collectively termed herein “anti-angiogenicAT3 products,” “anti-proliferative AT3 products,” aaAT, or aaAT3.

[0046] In addition to the sequences of AT3 described above, usefulnucleic acid molecules may comprise a nucleotide sequence which isgreater than about 80 percent, preferably greater than about 85 percent,more preferably greater than about 90 percent, and even more preferablygreater than about 95 percent, identical to the nucleotide sequences ofS-AT3, R-AT3 and L-AT3 deposited in GenBank. The substantially identicalsequence should, however, retain at least one of the activities ofinhibition of endothelial cell proliferation, inhibition of angiogenesisor inhibition of tumor growth (i.e., a biological equivalent).

[0047] To determine the percent identity of two nucleotide sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in the sequence of a first nucleotide sequence). Thenucleotides at corresponding nucleotide positions are then compared.When a position in the first sequence is occupied by the same nucleotideas the corresponding position in the second sequence, then the moleculesare identical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions×100).

[0048] The determination of percent identity between two sequences canbe accomplished Fusing a mathematical algorithm. A preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin et al., Proc.Mad. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm isincorporated into the NBLAST program which can be used to identifysequences having the desired identity to nucleotide sequences of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., Nucleic AcidsRes, 25:3389-3402 (1997). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,NBLAST) can be used. See http://www.ncbl.nlm.nih.gov. In one embodiment,parameters for sequence comparison can be set at W=12. Parameters canalso be varied (e.g., W=5 or W=20). The value “W” determines how manycontinuous nucleotides must be identical for the program to identify twosequences as containing regions of identity.

[0049] As appropriate, nucleic acid molecules of the present inventioncan be RNA, for example, mRNA, or DNA, such as cDNA and genomic DNA. DNAmolecules can be double-stranded or single-stranded; single stranded RNAor DNA can be either the coding, or sense, strand or the non-coding, orantisense, strand. Preferably, the nucleic acid molecule comprises atleast about 10 nucleotides, more preferably at least about 50nucleotides, and even more preferably at least about 200 nucleotides.The nucleic acid molecule can include all or a portion of the codingsequence of a gene and can further comprise additional non-codingsequences such as introns and non-coding 3′ and 5′ sequences (includingregulatory sequences, for example). Additionally, the nucleic acidmolecule can be fused to a marker sequence, for example, a sequencewhich encodes a polypeptide to assist in isolation or purification ofthe polypeptide. Such sequences include, but are not limited to, thosewhich encode a glutathione-S-transferase (GST) fusion protein and thosewhich encode a hemaglutin A (HA) polypeptide marker from influenza.

[0050] As used herein, an “isolated” gene or nucleic acid molecule isintended to mean a gene or nucleic acid molecule which is not flanked bynucleic acid molecules which normally (in nature) flank the gene ornucleic acid molecule (such as in genomic sequences) and/or has beencompletely or partially purified from other transcribed sequences (as ina cDNA or RNA library). For example, an isolated nucleic acid of theinvention may be substantially isolated with respect to the complexcellular milieu in which it naturally occurs. In some instances, theisolated material will form part of a composition (for example, a crudeextract containing other substances), buffer system or, reagent mix. Inother circumstance, the material may be purified to essentialhomogeneity, for example as determined by PAGE or column chromatographysuch as HPLC. Preferably, an isolated nucleic acid comprises at leastabout 50, 80 or 90 percent (on a molar basis) of all macromolecularspecies present. Thus, an isolated gene or nucleic acid molecule caninclude a gene or nucleic acid molecule which is synthesized chemicallyor by recombinant means. Recombinant DNA contained in a vector isincluded in the definition of “isolated” as used herein. Also, isolatednucleic acid molecules include recombinant DNA molecules in heterologoushost cells, as well as partially or substantially purified DNA moleculesin solution. In vivo and in vitro RNA transcripts of the DNA moleculesof the present invention are also encompassed by “isolated” nucleic acidmolecules. Such isolated nucleic acid molecules are useful in themanufacture of the encoded protein, as probes for isolating homologoussequences (e.g., from other mammalian species), for gene mapping (e.g.,by in situ hybridization with chromosomes), or for detecting expressionof the gene in tissue (e.g., human tissue) such as by Northern blotanalysis.

[0051] Thus, DNA molecules which comprise a sequence which is differentfrom the naturally-occurring nucleic acid molecule, but which, due tothe degeneracy of the genetic code, encode a substantially similarprotein or polypeptide are useful in this invention. The invention alsoencompasses variations of the nucleic acid molecules of the invention,such as those encoding portions, analogues or derivatives of the encodedprotein or polypeptide. Such variations can be naturally-occurring, suchas in the case of allelic variation, or non-naturally-occurring, such asthose induced by various mutagens and mutagenic processes. Intendedvariations include, but are not limited to, addition, deletion andsubstitution of one or more nucleotides which can result in conservativeor non-conservative amino acid changes, including additions anddeletions. Preferably, the nucleotide variations are silent; that is,they do not alter the characteristics or activity of the encoded proteinor polypeptide (i.e., a biological equivalent). As used herein,activities of the encoded protein or polypeptide include, but are notlimited to, inhibition of angiogenesis, inhibition of endothelial cellproliferation and inhibition of tumor growth. The invention alsoencompasses sequences that are not identical to AT3.

[0052] The invention also pertains to nucleic acid molecules whichhybridize under high stringency hybridization conditions (e.g., forselective hybridization) to a nucleotide sequence described herein.Hybridization probes are oligonucleotides which bind in a base-specificmanner to a complementary strand of nucleic acid. Such probes includepolypeptide nucleic acids, as described in Nielsen et al., Science 254,14971500 (1991). Such nucleic acid molecules can be detected and/orisolated by specific hybridization (e.g., under high stringencyconditions). “Stringency conditions” for hybridization is a term of artwhich refers to the incubation and wash conditions, e.g., conditions oftemperature and buffer concentration, which permit hybridization of aparticular nucleic acid to a second nucleic acid; the first nucleic acidmay be perfectly (i.e., 100%) complementary to the second, or the firstand second may share some degree of complementarity which is less thanperfect (e.g., 60%, 75%, 85%, 95%). For example, certain high stringencyconditions can be used which distinguish perfectly complementary nucleicacids from those of less complementarity. “High stringency conditions”,“moderate stringency conditions” and “low stringency conditions” fornucleic acid hybridizations are explained on pages 2.10.1-2.10.16 andpages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M.et al., “Current Protocols in Molecular Biology”, John Wiley & Sons,(1998)) the teachings of which are hereby incorporated by reference.Equivalent conditions can be determined by varying one or more of theparameters given as an example, as known in the art, while maintaining asimilar degree of identity or similarity between the target nucleic acidmolecule and the primer or probe used. Hybridizable nucleic acidmolecules are useful as probes and primers, e.g., for diagnosticapplications.

[0053] In addition to substantially full-length polypeptides encoded bynucleic acid molecules described herein, the present invention includesbiologically active fragments of the S-AT3, R-AT3 and L-AT3 biologicalequivalents, or analogs thereof, including organic molecules whichsimulate the interactions of S-AT3, R-AT3 or L-AT3. Biologically activefragments include any portion of the full-length polypeptide whichconfers a biological function on the variant gene product, includingligand binding and antibody binding, and particularly includinginhibition of endothelial cell proliferation, angiogenesis or tumorgrowth.

[0054] Also of use in the invention are fragments or portions of theisolated nucleic acid molecules described above. The term “fragment” isintended to encompass a portion of a nucleic acid molecule describedherein which is from at least about 7 contiguous nucleotides to at leastabout 25 contiguous nucleotides or longer in length. Such fragments areuseful as probes, e.g., for diagnostic methods, and also as primers. Thenucleotide sequences may also be an isolated portion of any of thenucleotide sequences of S-AT3, R-AT3 and L-AT3, which portion issufficient in length to distinctly characterize the sequence.Particularly preferred primers and probes selectively hybridize to thenucleotide sequences of S-AT3, R-AT3 and L-AT3. For example, fragmentswhich encode antigenic proteins or polypeptides described herein areuseful.

[0055] Also within the practice of the invention are anti-angiogenic AT3products and anti-proliferative AT3 products that are intended toencompass any fragments or conformations (e.g., the L conformation) ofAT3 which have anti-angiogenic and/or anti-proliferative activity,respectively. Anti-angiogenic activity and anti-proliferative activitycan be assessed according to methods described herein or according toother methods known in the art or may be any fragments or biologicalequivalents that mimic the active site.

[0056] The biological equivalents of AT3, may include, but are notlimited to, fragments of S-AT3, R-AT3, and L-AT3 that comprise theactive site; synthetic compounds that mimic the active site;conformational variations of other serpins; other conformations of AT3,aggregate forms and fusion proteins that exhibit anti-angiogenic andanti-proliferative properties. Conformational variations of otherserpins that may be useful in the practice of the invention include butare not limited to plasminogen activator inhibitor-1 (PAI-1), α2antiplasmin, α1 proteinase inhibitor, heparin cofactor II, C1 inhibitor,α1 antichymotrypsin, protease nexin 1, and pigment epithelial derivedfactor.

[0057] This invention also pertains to an isolated protein orpolypeptide encoded by the nucleic acid molecules of the invention. Theencoded proteins or polypeptides of the invention can be partially orsubstantially purified (e.g., purified to homogeneity), and/or aresubstantially free of other proteins. According to the invention, theamino acid sequence of the polypeptide can be that of thenaturally-occurring protein or can comprise alterations therein. Suchalterations include conservative or non-conservative amino acidsubstitutions, additions and deletions of one or more amino acids;however, such alterations should preserve at least one activity of theencoded protein or polypeptide, i.e., the altered or mutant proteinshould be a biological equivalent of the naturally-occurring protein.The mutation(s) should preferably preserve the endothelial cellproliferative inhibition, angiogenesis inhibition or tumor growthinhibition activities of the native protein or polypeptide. The presenceor absence of biological activity or activities can be determined byvarious functional assays as described herein. For example,glycosylation variants of AT3, along with β-AT3 (Olson et al., Archivesof Biochemistry and Biophysics 341(2):212-221 (1997)) are within thescope of the biological equivalents of AT3. The β-AT3 form may also beuseful as described herein.

[0058] Moreover, amino acids which are essential for the function of theencoded protein or polypeptide can be identified by methods known in theart. Particularly useful methods include identification of conservedamino acids in the family or subfamily, site-directed mutagenesis andalanine-scanning mutagenesis (for example, Cunningham and Wells, Science244:1081-1085 (1989)), crystallization and nuclear magnetic resonance.The altered polypeptides produced by these methods can be tested forparticular biologic activities, including immunogenicity andantigenicity.

[0059] Specifically, appropriate amino acid alterations can be made onthe basis of several criteria, including hydrophobicity, basic or acidiccharacter, charge, polarity, size, the presence or absence of afunctional group (e.g., —SH or a glycosylation site), and aromaticcharacter. Assignment of various amino acids to similar groups based onthe properties above will be readily apparent to the skilled artisan;further appropriate amino acid changes can also be found in Bowie etal., Science 247:1306-1310(1990).

[0060] For example, conservative amino acid replacements can be thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids are generally divided intofour families: (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine, histidine; (3) nonpolar—alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar—glycine, asparagine, glutamine, cystine, serine, threonine,tyrosine. Phenylalanine, tryptophan and tyrosine are sometimesclassified jointly as aromatic amino acids. For example, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine or a similar conservative replacement of an amino acid with astructurally related amino acid will not have a major effect on activityor functionality.

[0061] The polypeptides of the present invention can be used to raiseantibodies or to elicit an immune response. The polypeptides can also beused as a reagent, e.g., a labeled reagent, in assays to quantitativelydetermine levels of the protein or a molecule to which it binds (e.g., areceptor or a ligand) in biological fluids. The polypeptides can also beused as markers for tissues in which the corresponding protein ispreferentially expressed, either constitutively, during tissuedifferentiation, or in a diseased state. The polypeptides can be used toisolate a corresponding binding partner, e.g., receptor or ligand, suchas, for example, in an interaction trap assay, and to screen for peptideor small molecule antagonists or agonists.

[0062] The present invention also relates to antibodies which bind apolypeptide or protein of the invention. For instance, polyclonal andmonoclonal antibodies, including non-human and human antibodies,humanized antibodies, chimeric antibodies and antigen-binding fragmentsthereof (Current Protocols in Immunology, John Wiley & Sons, N.Y.(1994); EP Application 173,494 (Morrison); International PatentApplication W086/01533 (Neuberger); and U.S. Pat. No. 5,225,539(Winters)) which bind to the described S-AT3, R-AT3 or L-AT3 proteins orpolypeptides are within the scope of the invention. A mammal, such as amouse, rat, hamster or rabbit, can be immunized with an immunogenic formof the protein (e.g., the full length protein or a polypeptidecomprising an antigenic fragment of the protein which is capable ofeliciting an antibody response). Techniques for conferringimmunogenicity on a protein or polypeptide include conjugation tocarriers or other techniques well known in the art. The protein orpolypeptide can be administered in the presence of an adjuvant. Theprogress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassays can beused with the immunogen as antigen to assess the levels of antibody.

[0063] As described herein, AT3 and/or a fragment, conformation,biological equivalent, or derivative can be made or isolated by numerousmethods known in the art, including, but not limited to, purification,transgenic and recombinant methods.

[0064] The invention provides expression vectors containing a nucleicacid sequence described herein, operably linked to at least oneregulatory sequence. Many such vectors are commercially available, andother suitable vectors can be readily prepared by the skilled artisan.“Operably linked” or “operatively linked” is intended to mean that thenucleic acid molecule is linked to a regulatory sequence in a mannerwhich allows expression of the nucleic acid sequence. Regulatorysequences are art recognized and are selected to produce the encodedpolypeptide or protein. Accordingly, the term “regulatory sequence”includes promoters, enhancers, and other expression control elementswhich are described in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). For example,the native regulatory sequences or regulatory sequences native to thetransformed host cell can be employed. It should be understood that thedesign of the expression vector may depend on such factors as the choiceof the host cell to be transformed and/or the type of protein desired tobe expressed. For instance, the polypeptides of the present inventioncan be produced by ligating the cloned gene, or a portion thereof, intoa vector suitable for expression in either prokaryotic cells, eukaryoticcells or both (see, for example, Broach, et al., ExperimentalManipulation of Gene Expression, ed. M. Inouye (Academic Press, 1983) p.83; Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. Sambrook et al.(Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17).Typically, expression constructs will contain one or more selectablemarkers, including, but not limited to, the gene that encodesdihydrofolate reductase and the genes that confer resistance toneomycin, tetracycline, ampicillin, chloramphenicol, kanamycin andstreptomycin resistance.

[0065] Prokaryotic and eukaryotic host cells transfected by thedescribed vectors are also provided by this invention. For instance,cells which can be transfected with the vectors of the present inventioninclude, but are not limited to, bacterial cells such as E. coli (e.g.,E. coli K12 strains), Streptomyces, Pseudomonas, Serratia marcescens andSalmonella typhimurium, insect cells (baculovirus), includingDrosophila, fungal cells, such as yeast cells, plant cells and mammaliancells, such as thymocytes, Chinese hamster ovary cells (CHO), and COScells.

[0066] In one embodiment, at least one fragment, conformation,biological equivalent, or derivative of AT3 that is useful in thepractice of the invention is produced in vivo or ex vivo via genetherapy. For example, gene therapy may be used to produce AT3 or abiological equivalent. An enzyme that effectuates a conformationalchange in AT3 or a biological equivalent to an anti-angiogenic productis then delivered to the AT3 or the biological equivalent. Gene therapymay be used to produce an enzyme that effectuates a conformationalchange in AT3 or a biological equivalent to an anti-angiogenic product,or both an enzyme and AT3 or a biological equivalent. By using tissuespecific expression an anti-angiogenic product may be produced in vivoat a desired site.

[0067] Thus, a nucleic acid molecule described herein can be used toproduce a recombinant form of the protein via microbial or eukaryoticcellular processes. Ligating the polynucleic acid molecule into a geneconstruct, such as an expression vector, and transforming ortransfecting into hosts, either eukaryotic (yeast, avian, insect, plantor mammalian) or prokaryotic (bacterial cells), are standard proceduresused in producing other well known proteins. Similar procedures, ormodifications thereof, can be employed to prepare recombinant proteinsaccording to the present invention by microbial means or tissue-culturetechnology. Accordingly, the invention pertains to the production ofencoded proteins or polypeptides by recombinant technology.

[0068] The proteins and polypeptides of the present invention can beisolated or purified (e.g., to homogeneity) from recombinant cellculture by a variety of processes. These include, but are not limitedto, anion or cation exchange chromatography, ethanol precipitation,affinity chromatography and high performance liquid chromatography(HPLC). The particular method used will depend upon the properties ofthe polypeptide and the selection of the host cell; appropriate methodswill be readily apparent to those skilled in the art.

[0069] Following immunization, anti-peptide antisera can be obtained,and if desired, polyclonal antibodies can be isolated from the serum.Monoclonal antibodies can also be produced by standard techniques whichare well known in the art (Kohler and Milstein, Nature 256:495-497(1975); Kozbar et al., Immunology Today 4:72 (1983); and Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 7796(1985)). The term “antibody” as used herein is intended to includefragments thereof, such as Fab and F(ab)2. Antibodies described hereincan be used to inhibit the activity of the polypeptides and proteinsdescribed herein, particularly in vitro and in cell extracts, usingmethods known in the art. Additionally, such antibodies, in conjunctionwith a label, such as a radioactive label, can be used to assay for thepresence of the expressed protein in a cell from, e.g., a tissue sample,and can be used in an immunoabsorption process, such as an ELISA, toisolate the protein or polypeptide. Tissue samples which can be assayedinclude human tissues, e.g., differentiated and non-differentiatedcells, such as tumor cells. These antibodies are useful in diagnosticassays, or as an active ingredient in a pharmaceutical composition. Forexample, passive antibody therapy using antibodies which specificallybind S-AT3, R-AT3 or L-AT3 can be used to modulate (inhibit or enhance)endothelial cell proliferative- or angiogenic-dependent processes suchas reproduction, wound healing and tissue repair.

[0070] The present invention also encompasses the detection ofconformations including but not limited to S-AT3, R-AT3, and L-AT3,fragments, derivatives, conformational variations of other serpins, andbiological equivalents of AT3 in bodily fluids to determine thediagnosis or prognosis of endothelial cell proliferation related orangiogenesis-related disorders. As used herein, angiogenesis-relateddisorders include, but are not limited to, cancers, solid tumors, bloodborn tumors such as leukemias, tumor metastasis, benign tumors such ashemangiomas, acoustic neuromas, neurofibromas, trachomas and pyogenicgranulomas, rheumatoid arthritis, psoriasis, ocular anglogenic diseasessuch as diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia and rubeosis, Osler-Webber Syndrome, myocardialangiogenesis, plaque neovascularization, telangiectasia, hemophiliacjoints, angiofibroma and wound granulation. As used herein, endothelialcell proliferation-related disorders include, but are not limited to,intestinal adhesions, atherosclerosis, scleroderma and hypertrophicscars. Compounds described herein can also be used as birth controlagents by preventing the neovascularization required for embryoimplantation.

[0071] The invention also relates to a kit for detecting the presence offragments, conformations, derivatives, and biological equivalents ofAT3, including S-AT3, R-AT3 or L-AT3 in bodily fluids. Typically, thekit will comprise primary reagents (e.g., antibodies) capable ofdetecting the presence of fragments, conformations, derivatives, andbiological equivalents in a sample. The kit may also comprise adjunctreagents suitable for detecting binding of the primary reagent to thetarget.

[0072] The present invention also pertains to pharmaceuticalcompositions comprising fragments, conformations, derivatives, andbiological equivalents of AT3 including polypeptides and other compoundsdescribed herein. For instance, a polypeptide or protein, or prodrugthereof, of the present invention can be formulated with aphysiologically acceptable medium to prepare a pharmaceuticalcomposition. In one embodiment, an anti-angiogenic pharmaceuticalcomposition comprises a purified form of AT3 that reduces angiogenesis.In a preferred embodiment the purified form of AT3 is the L form or Rform of AT3 or a fragment or sequence which includes the active site orregion of the L form or R form of AT3. The particular physiologicalmedium may include, but is not limited to, water, buffered saline,polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol)and dextrose solutions. The optimum concentration of the activeingredient(s) in the chosen medium can be determined empirically,according to well known procedures, and will depend on the ultimatepharmaceutical formulation desired. Formulation of an agent to beadministered will vary according to the route of administration selected(e.g., solution, emulsion, capsule). An appropriate compositioncomprising the agent to be administered can be prepared in aphysiologically acceptable vehicle or carrier. For solutions oremulsions, suitable carriers include, for example, aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles can include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils, for instance. Intravenous vehicles can includevarious additives, preservatives, or fluid, nutrient or electrolytereplenishers and the like (See, generally, Remington's PharmaceuticalSciences, 17^(th) Edition, Mack Publishing Co., Pa., 1985). Forinhalation, the agent can be solubilized and loaded into a suitabledispenser for administration (e.g., an atomizer, nebulizer orpressurized aerosol dispenser).

[0073] The pharmaceutical compositions of the present invention may alsocomprise a composition that effectuates a conformational change in aserpin or produces a fragment, conformation, derivative, and biologicalequivalent of AT3 in vivo, for example, by the delivery of an enzyme.

[0074] Methods of introduction at the site of treatment include, but arenot limited to, intradermal, intramuscular, intra peritoneal,intravenous, rectal, vaginal, intra ocular, topical, subcutaneous, oraland intra nasal. Other suitable methods of introduction can also includegene therapy, rechargeable or biodegradable devices, viral vectors,naked DNA, lipids and slow release polymeric devices. The pharmaceuticalcompositions of this invention can also be administered as part of acombinatorial therapy with other agents. Nucleic acid sequences of theinvention can be used in gene therapy and introduced either in vivo orex vivo into cells for expression in a mammalian subject. Cells can alsobe cultured ex vivo in the presence of proteins of the present inventionin order to produce a desired effect on such cells. Treated cells canthen be introduced in vivo for therapeutic purposes.

[0075] The invention can be used to treat a variety of animals. Suitableanimals as used herein include mammals, including, but not limited to,primates (e.g., humans), dogs, cats, cows, horses, pigs, sheep, goatsand rodents (e.g., rats, mice and hamsters). Appropriate dosages (e.g.,those containing an “effective amount”) of a fragment, conformation,derivative or biological equivalent of AT3 will depend upon the physicalcharacteristics of the animal to be treated and on the disorder and(progression thereof) to be treated. One of ordinary skill in the artwould readily be able to determine what would be an effective amount.The agent can be administered alone or in combination with other agentsor treatment regimes, including chemotherapy and radiation. The agentcan be administered in multiple or single administrations providedsequentially or simultaneously.

[0076] It is also a subject of this invention to provide a new methodfor identifying an inhibitor of tumor growth or an agent that reducestumor growth. Prior methods typically required that two separate tumorinoculations or implants be carried out, the second being performedsubstantially later than the first. The reason for this approach wasthat it was thought that if the second tumor implant occurred at thesame time, or shortly after, the primary tumor was implanted, theprimary tumor would not be able to suppress the secondary tumor, becauseby the time the primary tumor reached sufficient size to release theinhibitors continuously into the circulation, angiogenesis in thesecondary tumor would already be well underway. Similarly, it was alsobelieved that if two tumors were implanted simultaneously (e.g., inopposite flanks), the inhibitors would have an equivalent inhibitingeffect on each other.

[0077] Work described herein has shown that tumor implants orinoculations can in fact be carried out substantially simultaneously inan animal in an effort to identify inhibitors of tumor growth. As usedherein, “substantially simultaneously” means that the tumor implants orinoculations occur at the same time or closely in sequence; there is norequirement that one tumor be implanted or inoculated first and allowedto grow to a particular size. The method of this invention thereforeprovides a substantial reduction in the time necessary to carry out thescreening, as it is not necessary to wait for the first tumor to growbefore implanting the second.

[0078] According to the method of the invention, an animal (e.g., amammal such as a mouse, rat, guinea pig or primate) is inoculated withan appropriate inoculum of tumor cells in each of two suitableinoculation sites substantially simultaneously. An appropriate inoculumof tumor cells is an amount of tumor cells sufficient to cause formationof a tumor, and is intended to include implantation of preformed tumorssuch as micrometastasis. The tumor cells can be derived from any tumors;for example, the tumors can include, but are not limited to, small celllung cancers and hepatocellular carcinomas. Appropriate inoculationsites include, but are not limited to, the subcutaneous space, thecornea, the lung, the breast, the prostate, the testes and the brain. Ina preferred embodiment, the inoculation sites are the flanks of theanimal.

[0079] The tumors are allowed to grow in the animal, and inhibition ofgrowth of one tumor, known as the subordinate tumor, with concomitantgrowth of the other tumor, known as the dominant tumor, is identified.Tumor size can be measured using methods known in the art, such as bymeasuring the diameter of the tumor using calipers. Using methodsdescribed herein and known in the art, such as selective in vivopassaging, dominant tumors can be selected which substantiallycompletely inhibit the growth of the subordinate tumor. As used herein,“substantially completely” is intended to include greater than about 80%inhibition of growth of the subordinate tumor. In a preferredembodiment, the inhibition is greater than about 90%, and in aparticularly preferred embodiment, the inhibition is near 100%. However,dominant tumors which inhibit the growth of the subordinate tumor to anydegree are useful.

[0080] Once a suitable dominant tumor is identified, the component(s)which inhibits tumor growth can be purified from the tumor. For example,the tumor can be grown in vitro and the component can be purified fromconditioned media from the tumor cells using methods described herein orother methods known in the art. With respect to protein or polypeptideidentification, bands identified by gel analysis can be isolated andpurified by HPLC, and the resulting purified protein can be sequenced.Alternatively, the purified protein can be enzymatically digested bymethods known in the art to produce polypeptide fragments which can besequenced. The sequencing can be performed, for example, by the methodsof Wilm et al. Nature 379(6564):466-469 (1996). The protein may beisolated by conventional means of protein biochemistry and purificationto obtain a substantially pure product, i.e., 80, 95 or 99% free of cellcomponent contaminants, as described in Jacoby, Methods in EnzymologyVolume 104, Academic Press, New York (1984); Scopes, ProteinPurification, Principles and Practice, 2nd Edition, Springer-Verlag, NewYork (1987); and Deutscher (ed), Guide to Protein Purification, Methodsin Enzymology, Vol. 182 (1990). If the protein is secreted, it can beisolated from the supernatant in which the host cell is grown. If notsecreted, the protein can be isolated from a lysate of the host cells.

[0081] Potential inhibitors can be tested in endothelial cellproliferation assays and/or angiogenesis assays (e.g., a CAM assay) toidentify inhibitors of endothelial cell proliferation and/orangiogenesis. For example, as described herein, bovine and human R-AT3and L-AT3 were identified as inhibiting endothelial cell proliferation,angiogenesis and tumor growth.

[0082] Compounds identified by this method as inhibiting endothelialcell proliferation, angiogenesis and/or tumor growth can be used in bothin vitro and in vivo methods to inhibit endothelial cell proliferation,angiogenesis and/or tumor growth as described herein for AT3.Furthermore, antagonists of identified compounds can be identified usingart recognized methods. For example, an antagonist to be tested can becombined with the compound in an endothelial cell proliferation assay orangiogenesis assay, and the level of endothelial cell proliferation orangiogenesis can be assessed relative to the results in the absence ofthe putative antagonist. Antagonists can be nucleic acids, proteins orpolypeptides, small biologically active molecules, or large cellularstructures and can be used to enhance endothelial cell proliferationand/or angiogenesis, such as in wound healing. Use of compoundsidentified by this method for inhibition or enhancement of endothelialcell proliferation and/or angiogenesis and for inhibition of tumorgrowth, as well as novel compounds identified by this method, are withinthe scope of the invention.

[0083] The present invention will now be illustrated by the followingExamples, which are not intended to be limiting in any way. Theteachings of all references cited herein are incorporated herein byreference in their entirety.

EXAMPLES Example 1

[0084] Purification of Bovine and Human Antithrombin III

[0085] Bovine calf serum was thawed and heat- inactivated (56C×20minutes) and then stored at 4° C. for 14-21 days to allow fordegradation of AT3 to the R form. Serum was diluted 3-fold with 10 mMTris pH 7 and then applied to a CM Sepharose column (5×35 cm) coupled toa DEAE Sepharose column (5×35 cm) after equilibration with 10 mM Tris pH7. Both columns were washed extensively with 10 mM Tris pH 7 and thenuncoupled. The DEAE column was washed extensively with 50 mM NaCl in 10m-M Tris and then coupled to a heparin Sepharose column (2.5×3 5 cm)which was equilibrated with 0.2 M NaCl 10 mM Tris pH 7. Bound proteinfrom the DEAE column was eluted directly onto the heparin Sepharosecolumn using 0.2 M NaCl 10 mM Tris pH 7 and the columns were uncoupled.The heparin Sepharose column was washed extensively with 0.5 M NaCl andthen eluted with a continuous gradient of 0.6-2 M NaCl (550 ml totalvolume) followed by an additional 250 ml of 2 M NaCl. Fractions werecollected and an aliquot of each was tested on capillary endothelialcells. Fractions that inhibited were pooled and concentrated using aNanoSpin 30K centrifugal concentrator.

[0086] Human plasma was centrifuged (10,000 rpm×30 minutes), filtered(0.45 μm), and diluted 3-fold with 10 mM Tris pH 7. The diluted plasmawas then applied to a CM Sepharose column (5×35 cm) coupled to a DEAESepharose column (5×35 cm) after equilibration with 10 mM Tris pH 7.Both columns were washed extensively with 10 mM Tris pH 7 and thenuncoupled. The DEAE column was washed extensively with 50 mm NaCl in 10mM Tris and then coupled to a heparin Sepharose column (2.5×35 cm) whichwere equilibrated with 0.2 M NaCl 10 mM Tris pH 7. Bound protein fromthe DEAE column was eluted directly onto the heparin Sepharose columnusing 0.2 M NaCl 10 mM Tris pH 7 and the columns were uncoupled. Theheparin Sepharose column was washed extensively with PBS followed by 0.5M NaCl in 10 mM Tris pH 7 and then eluted with a continuous gradient of0.6-2 M NaCl (550 ml total volume) followed by an additional 250 ml of 2M NaCl. Purified intact native AT3 was cleaved with porcine pancreaticelastase to produce the R-conformation incubated at 4° C. in 0.9 Mguanidine and then dialyzed against PBS to produce the L-conformation.Purity of the final samples were assessed by SDS-PAGE with silverstaining. Protein concentration was determined using a Biorad assay.

Example 2

[0087] Production of R and L forms of Antithrombin III

[0088] Intact bovine and human AT3 were obtained from Sigma orCalbiochem, respectively, or from human plasma as described above. Humanand bovine intact AT3 were incubated in 0.9 M guanidium chloride toproduce the stable locked conformation (L form) of the molecule asdescribed previously by Carrell et al. After 12 hours of incubation,guanidium chloride was removed by dialysis (30K membrane) in PBS andsamples were concentrated. Alternatively, AT3 was cleaved withpancreatic elastase as described. In order to obtain complete cleavage,the method was modified and AT3 was incubated with elastase at a 1:5molar ratio for 12 hours at 37° C. The reaction was quenched by applyingthe mixture to a heparin Sepharose column at 4° C. The cleaved AT3 waspurified to homogeneity using heparin Sepharose and then concentratedusing a Nanospin 30K centrifugal concentrator.

Example 3

[0089] Inhibitory Activity on Endothelial Proliferation

[0090] To determine if the inhibitory activity on endothelial cellproliferation of AT3 was ispecific, AT3 from bovine and human sourceswas screened on a panel of nonendothelial cell lines in vitro, asspecificity in vitro may predict for lack of toxicity in vivo. Of allthe cell types screened, only capillary endothelial cells weresignificantly inhibited even at log fold higher doses. The samespecificity was seen for the L- and R forms of human and bovine AT3. Theintact native (S-AT3) molecule had no significant effect onnon-endothelial cells and only marginally inhibited capillaryendothelial cells at doses in excess of 10 ug/ml.

[0091] Purified intact bovine and human AT3 (S-AT3), R-AT3 and L-AT3were tested on capillary endothelial cells in a proliferation assay(FIGS. 3 and 4, respectively). The L-AT3 potently inhibited capillaryendothelial cell proliferation in a dose dependent and reversiblefashion with half maximal inhibition seen at approximately 50 ng/ml forboth the bovine and human protein (FIGS. 3 and 4, respectively). Theinhibition was comparable for that seen with the cleaved form of AT3from the H69 conditioned media or from a digestion of the intact proteinwith pancreatic elastase. The intact native conformation of bovine andhuman AT3 had no effect on capillary endothelial cell proliferation atcomparable doses (FIGS. 3 and 4, respectively) but did show marginalinhibition at doses in excess of 5 μg/ml. It was determined thatvirtually all of the AT3 was in the cleaved (R) form that inhibitedendothelial cell proliferation in a dose dependent fashion (FIGS. 3 and4). The data demonstrates that the conformational change that occursafter cleavage of the AT3 molecule confers antiangiogenic activity. Theterm aaAT3 is used to describe the antiangiogenic form of AT3.

Example 4

[0092] Inhibition of in vivo Angiogenesis

[0093] In order to determine whether the AT3 fragment, R-conformation,or L-conformation could inhibit in vivo angiogenesis, the chickchorioallantoic membrane (CAM) assay was used. CAM-Assay

[0094] Three-day-old, fertilized white Leghorn eggs (Spafas, Norwich,Conn.) were cracked, and embryos with intact yolks were placed in 100×20mm petri dishes. After three days of incubation (37° C. and 3% CO₂), amethylcellulose disc containing AT3 was applied to the CAM of individualembryos. The discs were made by desiccation of AT3 in 10 μl of 0.45%methylcellulose on teflon rods. After 48 hours of incubation, embryosand CAMs were observed by means of a stereomicroscope. Embryos wereobserved daily until there was no evidence of inhibitory zones.

[0095] Intact native AT3 had no effect on angiogenesis in the assay butdid cause local bleeding at the injection site at higher doses. Incontrast, both the R- and L forms of human and bovine AT3 potentlyinhibited angiogenesis at doses of 20 ug per CAM without evidence ofbleeding. In all embryos tested with two separate batches of AT3, therewas a potent and sustained inhibition of angiogenesis. No hemorrhage wasseen in any of the treated groups, consistent with prior reports thatdemonstrate these conformations do not bind or inhibit thrombin. Therewas no evidence of any toxic or inflammatory reaction from any of theproteins tested in the assay at any dose.

Example 5

[0096] Inhibition of Tumor Growth

[0097] Treatment of Human Malignant Neuroblastoma

[0098] Male 6-8 week old C57BI6/J (Jackson Labs, Bar Harbor, Me.) orSCID (MGH) mice were used. Mice were acclimated, caged in groups of 4 orless, their backs shaved, and fed a diet of animal chow and water adlibitum. Methoxyfurane by inhalation was used for anesthesia andeuthanasia.

[0099] Immunocompromised SCID mice were implanted with humanneuroblastoma cells and tumors were allowed to grow to 1% of bodyweight. This tumor line (SK-NAS) has a consistent pattern of growth.Tumors were measured with a dial caliper, volumes determined using theformula width 2×length×0.52, and the ratio of the treated-to-controlvolume was determined for the last time point. When tumor volume reached179-200 mm³, mice were randomized into groups and treated with bovinecleaved AT3 (R) form or the cleaved (R), locked (L) or native intact (S)conformations of human AT3 or vehicle control injected into thesubcutaneous flanks once daily at a site distant from the tumor at adose of 25 mg/kg (500 μg per 20 gram mouse). The experiment wasterminated and mice sacrificed and autopsied when control mice began todie or experience significant morbidity.

[0100] The intact native form of AT3 had no effect on tumor growth ascompared to control mice treated with vehicle alone (FIG. 5). Incontrast, mice treated with the R- and L-conformation of AT3 had acomplete regression of the implanted neuroblastoma; tumors in these micepersisted as small barely visible subcutaneous nodules (FIG. 5). Notoxicity was seen in any of the treated mice except for some localbleeding at the injection site of the mice treated with the nativeintact AT3.

[0101] In a separate experiment, SCID mice implanted with humanneuroblastoma were treated with latent or locked human AT3 at a dose of15 mg/kg/day (FIG. 6) and both potently inhibited tumor growth. Thesedata show that aaAT3 is a potent inhibitor of angiogenesis and tumorgrowth.

Example 6

[0102] Method of Identifying Inhibitors of Angiogenesis, EndothelialCell Proliferation, and/or Tumor Growth

[0103] In order for a carcinoma to expand beyond a microscopicprevascular state, it must produce stimulators of angiogenesis in excessof inhibitors and must sustain this imbalance. It has been demonstratedthat the continued production of angiogenesis inhibitors provides amechanism for the inhibition of tumor growth by tumor mass. Using murinemodels of concomitant resistance, angiostatin from a murine lungcarcinoma and endostatin from a murine hemangioendothelioma wereidentified. To determine if human tumors might also produce inhibitorsof angiogenesis, human small cell lung cancer was screened for theability of a primary tumor on the flank of an immunocompromised mouse toinhibit the growth of a similar implant on the opposite flank. Smallcell lung cancer was chosen because, clinically, metastatic small celllung cancer often grows rapidly after definitive treatment of theprimary disease.

[0104] The phenomenon of the rapid growth of metastasis has beenreferred to as concomitant immunity, which could be due to theproduction of an inhibitor of angiogenesis by small cell lung cancer.Several human small cell lung cancer cell lines were screened for theability of a primary tumor on the flank of an immunocompromised mouse toinhibit the growth of a similar tumor on the opposite flank. One of thetumor lines obtained, a variant of NCI-H69 obtained from the ATCC, whichwas originally derived from a primary tumor, inhibited a similar tumorby over 80%. By selective in vivo passage, two variants of the H69 linewere developed (H69i and H69ni). A tumor model was developed using H69iin which the inhibition of one tumor by another was virtually 100% (lineH69i) [FIG. 7A]. A second variant was also developed in which one tumordid not inhibit the other to a significant degree (H69ni) [FIG. 7B].

[0105] Tumor cell lines derived from the inhibitory and non-inhibitoryvariants of H69 were established in vitro. To screen for evidence of theproduction of an angiogenesis inhibitor, conditioned media was tested onbovine capillary endothelial cells in a 72-hour proliferation assay.When cells were nearly confluent, conditioned media was collected.Conditioned media from the H69i cells potently and reversibly inhibitedcapillary endothelial cell proliferation. Conditioned media from theH69ni cell line had no significant inhibitory effect on the endothelialcells. The data demonstrates that the purified inhibitor of endothelialcell proliferation generated by the H69i cells is at least in partresponsible for the concomitant resistance observed in the tumor model.

[0106] Collection of Conditioned Media and Cell Culture

[0107] Human small cell lung carcinoma cell lines were obtained from theATCC and maintained in culture in DMEM supplemented with 10%heat-inactivated fetal calf serum and 1%glutamine-penicillin-streptomycin in a 37° C. and 10% CO₂ incubator.Optimal conditions were developed for conditioned media using theminimal volume of media supplemented with the least amount of serum andthe maximal contact time for cell viability. To produce conditionedmedia, 80 milliliter of DMEM with 2.5% FCS and 1% GPS was added to nearconfluent cells in 900 cm2 roller bottles. After 96 hours at 37° C. and10% CO₂, media was collected, centrifuged (10,000 rpm for 20 minutes),filtered (0.45 μm), and stored at 4° C. The cells were noted to grow asspheroids, which were loosely adherent to the plastic. Media wascollected every 96 hours until the spheroid density expanded beyond thelimits of the surface area of the roller bottle.

[0108] Purification of Inhibitory Activity from Conditioned Media

[0109] DEAE, CM, lysine, and heparin Sepharose, Sephacryl S200 HR gel,and a SynChropak RP-4 C4 reverse-phase column were all preparedaccording to the manufacturers' recommendations. Pooled conditionedmedia (3-3.5 liters) was diluted three-fold with 10 mM Tris pH 7 andapplied to a CM Sepharose column (5×35 cm) coupled to a DEAE Sepharosecolumn (5×35 cm) after equilibration with 10 mM Tris pH 7. Both columnswere washed extensively with 10 mM Tris pH 7 and then uncoupled. Eachcolumn was eluted with a step gradient of NaCl in 10 mM Tris pH 7 with50 mM, 0.2 M, 0.6 M, I M and 2 M steps. The DEAE column was washedextensively with 50 mM NaCl in 10 mM Tris to remove phenol red.Fractions with evidence of protein by A280 for each step were pooled andan aliquot of each was applied to bovine capillary endothelial cells ina 72-hour proliferation assay. The 0.2 M NaCl elution of the DEAE columnwas found to inhibit capillary endothelial cell proliferation and wasdiluted 2-fold with 10 mM Tris pH 7.

[0110] A heparin Sepharose column (2.5×35 cm) was equilibrated with 0.2M NaCl 10 mM Tris pH 7 and the inhibitory sample from the DEAE columnwas applied. The column was washed with phosphate buffered saline. Thecolumn was then eluted with a continuous gradient of 0.2-2 M NaCI (550ml total volume) followed by an additional 250 ml of 2 M NaCl. Fractionswere collected and an aliquot of each was tested on capillaryendothelial cells. Inhibitory activity was found in fractions eluting at1-1.2 M NaCl. Fractions that inhibited were pooled and concentrated to1.5 ml using a NanoSpin 30K centrifugal concentrator.

[0111] The concentrated sample was applied to a Sephacryl S200 HR column(1.5×75 cm) which was first equilibrated with PBS. The column was elutedwith PBS and an aliquot of each fraction collected was tested oncapillary endothelial cells. Fractions with inhibitory activity werepooled and concentrated to 1.5 ml using a NanoSpin centrifugalconcentrator.

[0112] A SynChropak RP-4 (4.6×100 mm) high performance liquidchromatography (HPLC) column was equilibrated with H₂0/0.1%trifluoroacetic acid (TFA) and HPLC-grade reagents (Pierce, Rockford,Ill.) were used. The sample from gel filtration was filtered (0.22 μm)and then applied to the column and the column washed with theequilibration buffer. The column was then eluted with a gradient ofacetonitrile in 0.1% TFA at 0.5 m/min and I ml fractions were collected.An aliquot of each fraction was evaporated by vacuum centrifugation,resuspended in PBS, and applied to capillary endothelial cells. Theinhibitory activity was further purified to apparent homogeneity bysubsequent cycles on the C4 column.

[0113] Fractions containing inhibitory activity evaluated by SDS-PAGEand the activity was associated with a band of apparent reducedmolecular weight (Mr) of 55 kDa that copurified with a 58-60 kDa band(FIG. 8b). The inhibitory activity was associated with the 55 kDa band.The 55 kDa band was purified to homogeneity using a C4 reverse phaseHPLC column and eluted at 55% acetonitrile in 0.1% trifluoroacetic acid(FIG. 8a).

[0114] The inhibitory fraction from the final HPLC run was analyzed bymicrosequence analysis. Protein Microsequencing

[0115] The 55 kDa (reduced) inhibitor of capillary endothelial cellproliferation was purified to homogeneity from batches of conditionedmedia. After the final HPLC, a sample containing a single 55 kDa bandafter staining with silver was used for N-terminal sequence analysis.The N-terminal sequence was determined by automated Edman degradation ona PE/ABD Model 470A protein sequencer (Foster City, Calif.) operatedwith gas-phase delivery of trifluoroacetic acid. Sequence librarysearches and alignments were performed against combined GenBank,Brookhaven Protein, SWISS-PROT, and PIR databases. Searches wereperformed at the National Center for Biotechnology Information throughthe use of the BLAST network service.

[0116] Sequence analysis revealed identity to bovine AT3 (FIG. 2). Massspectroscopy was performed and revealed a molecular weight of 50 kd.These data identified the inhibitor of angiogenesis as the cleaved form(R-conformation) of bovine AT3 (FIG. 2). SDS-PAGE behavior is also datawhich tends to confirm the R confirmation. The cleavage site betweenSer₃₈₆ and Thre₃₈₇ for bovine AT3 has not previously been described andsuggests that a novel enzyme may be involved. Other enzymes that cleaveAT3 include thrombin (Arg₃₉₄-Ser₃₉₅), pancreatic elastase(Val₃₈₈-Iso₃₈₉), human neutrophil elastase (Iso₃₉₁-Ala₃₉₂), and a numberof others known in the art.

Example 7

[0117] Purification of Bovine aaAT3 from BxPC3 Conditioned Media

[0118] B×PC3 conditioned media (5% FCS) was applied to heparin-Sepharosecolumn, previously equilibrated with 50 mM Tris-HCl, pH 7.4. The columnwas washed with 2-3 column volumes, and protein was eluted withincremental 0.5M NaCl steps (3 column volumes). Fractions were assayedfor the ability to inhibit the proliferation of endothelial cells usingwell established assay techniques. The fraction eluting fromheparin-Sepharose between 1-1.5M NaCl contained a 58kDa protein thatinhibited endothelial cell proliferation. This fraction was concentratedby membrane filtration and applied to a Superdex200 gel filtrationcolumn. Fractions were assayed for the ability to inhibit endothelialcell proliferation, and a fraction containing a 58kDa protein wasidentified. Sequence analysis of this single band determined it to bebovine antithrombin. Subsequent biochemcial analysis indicated that thisAT molecule, with anti-endothelial function, was in fact a “latent” formof bovine AT produced specifically by the B×PC3 cells. This moleculeinhibits the proliferation and migration of endothelial cells in a dosedependent manner.

[0119] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1 1 1 464 PRT Homo sapiens Antithrombin III 1 Met Tyr Ser Asn Val IleGly Thr Val Thr Ser Gly Lys Arg Lys Val 1 5 10 15 Tyr Leu Leu Ser LeuLeu Leu Ile Gly Phe Trp Asp Cys Val Thr Cys 20 25 30 His Gly Ser Pro ValAsp Ile Cys Thr Ala Lys Pro Arg Asp Ile Pro 35 40 45 Met Asn Pro Met CysIle Tyr Arg Ser Pro Glu Lys Lys Ala Thr Glu 50 55 60 Asp Glu Gly Ser GluGln Lys Ile Pro Glu Ala Thr Asn Arg Arg Val 65 70 75 80 Trp Glu Leu SerLys Ala Asn Ser Arg Phe Ala Thr Thr Phe Tyr Gln 85 90 95 His Leu Ala AspSer Lys Asn Asp Asn Asp Asn Ile Phe Leu Ser Pro 100 105 110 Leu Ser IleSer Thr Ala Phe Ala Met Thr Lys Leu Gly Ala Cys Asn 115 120 125 Asp ThrLeu Gln Gln Leu Met Glu Val Phe Lys Phe Asp Thr Ile Ser 130 135 140 GluLys Thr Ser Asp Gln Ile His Phe Phe Phe Ala Lys Leu Asn Cys 145 150 155160 Arg Leu Tyr Arg Lys Ala Asn Lys Ser Ser Lys Leu Val Ser Ala Asn 165170 175 Arg Leu Phe Gly Asp Lys Ser Leu Thr Phe Asn Glu Thr Tyr Gln Asp180 185 190 Ile Ser Glu Leu Val Tyr Gly Ala Lys Leu Gln Pro Leu Asp PheLys 195 200 205 Glu Asn Ala Glu Gln Ser Arg Ala Ala Ile Asn Lys Trp ValSer Asn 210 215 220 Lys Thr Glu Gly Arg Ile Thr Asp Val Ile Pro Ser GluAla Ile Asn 225 230 235 240 Glu Leu Thr Val Leu Val Leu Val Asn Thr IleTyr Phe Lys Gly Leu 245 250 255 Trp Lys Ser Lys Phe Ser Pro Glu Asn ThrArg Lys Glu Leu Phe Tyr 260 265 270 Lys Ala Asp Gly Glu Ser Cys Ser AlaSer Met Met Tyr Gln Glu Gly 275 280 285 Lys Phe Arg Tyr Arg Arg Val AlaGlu Gly Thr Gln Val Leu Glu Leu 290 295 300 Pro Phe Lys Gly Asp Asp IleThr Met Val Leu Ile Leu Pro Lys Pro 305 310 315 320 Glu Lys Ser Leu AlaLys Val Glu Lys Glu Leu Thr Pro Glu Val Leu 325 330 335 Gln Glu Trp LeuAsp Glu Leu Glu Glu Met Met Leu Val Val His Met 340 345 350 Pro Arg PheArg Ile Glu Asp Gly Phe Ser Leu Lys Glu Gln Leu Gln 355 360 365 Asp MetGly Leu Val Asp Leu Phe Ser Pro Glu Lys Ser Lys Leu Pro 370 375 380 GlyIle Val Ala Glu Gly Arg Asp Asp Leu Tyr Val Ser Asp Ala Phe 385 390 395400 His Lys Ala Phe Leu Glu Val Asn Glu Glu Gly Ser Glu Ala Ala Ala 405410 415 Ser Thr Ala Val Val Ile Ala Gly Arg Ser Leu Asn Pro Asn Arg Val420 425 430 Thr Phe Lys Ala Asn Arg Pro Phe Leu Val Phe Ile Arg Glu ValPro 435 440 445 Leu Asn Thr Ile Ile Phe Met Gly Arg Val Ala Asn Pro CysVal Lys 450 455 460

We claim:
 1. A method of reducing angiogenesis in a mammal comprisingdelivering to said mammal a composition comprising at least onefragment, conformation, biological equivalent, or derivative ofantithrombin III wherein said fragment, conformation, biologicalequivalent, or derivative of antithrombin III reduces angiogenesis.
 2. Amethod of reducing angiogenesis in a mammal according to claim 1 whereinsaid composition further comprises a physiologically acceptable vehicle.3. A method of reducing angiogenesis in a mammal according to claim 1wherein said at least one fragment, conformation, biological equivalent,or derivative of antithrombin III is chosen from the L form ofantithrombin III and the R form of antithrombin III.
 4. A method ofreducing angiogenesis in a mammal according to claim 1 wherein said atleast one fragment, conformation, biological equivalent, or derivativeof antithrombin III is chosen from a synthesized fragment ofantithrombin III that reduces angiogenesis, a conformational variationof serpins that reduce angiogenesis, an aggregate form of antithrombinIII that reduces angiogenesis, and a fusion protein of antithrombin IIIthat reduces angiogenesis.
 5. A method of reducing angiogenesis in amammal according to claim 1 wherein said conformational variation ofserpins is chosen from conformational variations of plasminogenactivator inhibitor-1, α₂ antiplasmin, α1 proteinase inhibitor, heparincofactor II, C1 inhibitor, α1 antichymotrypsin, protease nexin 1 orpigment epithelial derived factor.
 6. A method of reducing angiogenesisin a mammal according to claim 1 wherein said at least one fragment,conformation, biological equivalent, or derivative of antithrombin IIIis produced transgenically, recombinantly, or purified from mammalantithrombin III.
 7. A method of reducing angiogenesis in a mammalaccording to claim 1 wherein said at least one fragment, conformation,biological equivalent, or derivative of antithrombin III is produced invivo by the delivery of an enzyme.
 8. A method of reducing endothelialcell proliferation in a mammal comprising delivering to said mammal acomposition comprising at least one fragment, conformation, biologicalequivalent, or derivative of antithrombin III wherein said fragment,conformation, biological equivalent, or derivative of antithrombin IIIreduces endothelial cell proliferation.
 9. A method of reducingendothelial cell proliferation in a mammal according to claim 8 whereinsaid composition further comprises a physiologically acceptable vehicle.10. A method of reducing endothelial cell proliferation in a mammalaccording to claim 8 wherein said at least one fragment, conformation,biological equivalent, or derivative of antithrombin III is chosen fromthe L form of antithrombin III and the R form of antithrombin III.
 11. Amethod of reducing endothelial cell proliferation in a mammal accordingto claim 8 wherein said at least one fragment, conformation, biologicalequivalent, or derivative of antithrombin III is chosen from asynthesized fragment of antithrombin III that reduces endothelial cellproliferation, a conformational variation of serpins that reduceendothelial cell proliferation, an aggregate form of antithrombin IIIthat reduces endothelial cell proliferation, and a fusion protein ofantithrombin III that reduces endothelial cell proliferation.
 12. Amethod of reducing endothelial cell proliferation in a mammal accordingto claim 8 wherein said conformational variation of serpins is chosenfrom conformational variations of plasminogen activator inhibitor-1, α₂antiplasmin, α1 proteinase inhibitor, heparin cofactor II, C1 inhibitor,α1 antichymotrypsin, protease nexin 1 or pigment epithelial derivedfactor.
 13. A method of reducing endothelial cell proliferation in amammal according to claim 8 wherein said at least one fragment,conformation, biological equivalent, or derivative of antithrombin IIIis produced transgenically, recombinantly, or purified from mammalantithrombin III.
 14. A method of reducing endothelial cellproliferation in a mammal according to claim 8 wherein said at least onefragment, conformation, biological equivalent, or derivative ofantithrombin III is produced in vivo by the delivery an enzyme.
 15. Amethod of reducing tumor growth in a mammal comprising delivering tosaid mammal a composition comprising at least one fragment,conformation, biological equivalent, or derivative of antithrombin IIIwherein said fragment, conformation, biological equivalent, orderivative of antithrombin III reduces tumor growth.
 16. A method ofreducing tumor growth in a mammal according to claim 15 wherein saidcomposition further comprises a physiologically acceptable vehicle. 17.A method of reducing tumor growth in a mammal according to claim 15wherein said at least one fragment, conformation, biological equivalent,or derivative of antithrombin III is chosen from the L form ofantithrombin III and the R form of antithrombin III.
 18. A method ofreducing tumor growth in a mammal according to claim 15 wherein said atleast one fragment, conformation, biological equivalent, or derivativeof antithrombin III is chosen from a synthesized fragment ofantithrombin III that reduces endothelial cell proliferation, aconformational variation of serpins that reduce endothelial cellproliferation, an aggregate form of antithrombin III that reducesendothelial cell proliferation, and a fusion protein of antithrombin IIIthat reduces endothelial cell proliferation.
 19. A method of reducingtumor growth in a mammal according to claim 15 wherein saidconformational variation of serpins is chosen from conformationalvariations of plasminogen activator inhibitor-1, α₂ antiplasmin, α1proteinase inhibitor, heparin cofactor II, C1 inhibitor, α1antichymotrypsin, protease nexin 1 or pigment epithelial derived factor.20. A method of reducing tumor growth in a mammal according to claim 15wherein said at least one fragment, conformation, biological equivalent,or derivative of antithrombin III is produced transgenically,recombinantly, or purified from mammal antithrombin III.
 21. A method ofreducing tumor growth in a mammal according to claim 15 wherein said atleast one fragment, conformation, biological equivalent, or derivativeof antithrombin III is produced in vivo by the delivery of an enzyme.22. A method for identifying an agent that reduces tumor growth orangiogenesis comprising the steps of a) inoculating a mammal with aninoculum of tumor cells in each of two inoculation sites substantiallysimultaneously; b) identifying reduction of growth of a tumor at oneinoculation site, wherein said tumor at the one inoculation site is asubordinate tumor, with concomitant growth of a tumor at the otherinoculation site, wherein said tumor at the other inoculation site is adominant tumor; c) isolating cells from said dominant tumor; and d)purifying a component which reduces endothelial cell proliferation,angiogenesis or both endothelial cell proliferation and angiogenesisfrom the isolated dominant tumor cells.
 23. A method for identifying anagent that reduces tumor growth or angiogenesis according to claim 22wherein the tumor cells are derived from tumors selected from small celllung cancers and hepatocellular carcinomas.
 24. A method for identifyingan agent that reduces tumor growth or angiogenesis according to claim 22wherein the inoculation sites are the flanks of the mammal.
 25. A methodfor identifying an agent that reduces tumor growth or angiogenesisaccording to claim 22 wherein the component of step (d) is purified fromconditioned media from the cells of step (c).
 26. A method foridentifying an agent that reduces tumor growth or angiogenesis accordingto claim 22 wherein the agent that reduces tumor growth reducesendothelial cell proliferation.
 27. A method for identifying an agentthat reduces tumor growth or angiogenesis according to claim 22 whereinthe agent that reduces tumor growth reduces angiogenesis.
 28. A methodof reducing tumor growth comprising delivering to a mammal the agentthat reduces tumor growth or angiogenesis identified by the methodaccording to claim
 22. 29. A method of treating a disorder mediated byendothelial cell proliferation comprising delivering to a mammal acomposition comprising at least one fragment, conformation, biologicalequivalent, or derivative of antithrombin III wherein said fragment,conformation, biological equivalent, or derivative of antithrombin IIIis delivered in an amount effective to reduce endothelial cellproliferation.
 30. A method of treating a disorder mediated byendothelial cell proliferation according to claim 29 wherein said atleast one fragment, conformation, biological equivalent, or derivativeof antithrombin III is chosen from the L form of antithrombin III andthe R form of antithrombin III.
 31. A method of treating a disordermediated by endothelial cell proliferation according to claim 29 whereinsaid at least one fragment, conformation, biological equivalent, orderivative of antithrombin III is chosen from a synthesized fragment ofantithrombin III that reduces endothelial cell proliferation, aconformational variations of serpins that reduce endothelial cellproliferation, an aggregate form of antithrombin III that reducesendothelial cell proliferation, and a fusion protein of antithrombin IIIthat reduces endothelial cell proliferation.
 32. A method of treating adisorder mediated by endothelial cell proliferation according to claim29 wherein said conformational variation of serpins is chosen fromconformational variations of plasminogen activator inhibitor-1, α₂antiplasmin, α1 proteinase inhibitor, heparin cofactor II, C1 inhibitor,α1 antichymotrypsin, protease nexin 1 or pigment epithelial derivedfactor.
 33. A method of treating a disorder mediated by endothelial cellproliferation according to claim 29 wherein said at least one fragment,conformation, biological equivalent, or derivative of antithrombin IIIis produced transgenically, recombinantly, or purified from mammalantithrombin III.
 34. A method of treating a disorder mediated byangiogenesis comprising delivering to a mammal a composition comprisingat least one fragment, conformation, biological equivalent, orderivative of antithrombin III wherein said fragment, conformation,biological equivalent, or derivative of antithrombin III is delivered inan amount effective to reduce angiogenesis.
 35. A method of treating adisorder mediated by angiogenesis according to claim 34 wherein said atleast one fragment, conformation, biological equivalent, or derivativeof antithrombin III is chosen from the L form of antithrombin III andthe R form of antithrombin III.
 36. A method of treating a disordermediated by angiogenesis according to claim 34 wherein said at least onefragment, conformation, biological equivalent, or derivative ofantithrombin III is chosen from a synthesized fragment of antithrombinIII that reduces angiogenesis, a conformational variation of serpinsthat reduce angiogenesis, an aggregate form of antithrombin III thatreduces angiogenesis, and a fusion protein of antithrombin III thatreduces angiogenesis.
 37. A method of treating a disorder mediated byangiogenesis according to claim 34 wherein said conformational variationof serpins is chosen from conformational variations of plasminogenactivator inhibitor-1, α₂ antiplasmin, α1 proteinase inhibitor, heparincofactor II, C1 inhibitor, α1 antichymotrypsin, protease nexin 1 orpigment epithelial derived factor.
 38. A method of treating a disordermediated by angiogenesis according to claim 35 wherein said at least onefragment, conformation, biological equivalent, or derivative ofantithrombin III is produced transgenically, recombinantly, or purifiedfrom mammal antithrombin III.
 39. A method of enhancing angiogenesis orendothelial cell proliferation comprising delivering a compositioncomprising at least one antagonist of a fragment, conformation,biological equivalent, or derivative of antithrombin III to a mammalwherein said fragment, conformation, biological equivalent, orderivative of antithrombin III reduces angiogenesis.
 40. Aanti-angiogenic pharmaceutical composition comprising a purified form ofantithrombin III that reduces angiogenesis chosen from the (R) form ofantithrombin III or the (L) form of antithrombin III, and aphysiologically acceptable carrier.